A major enigma in the field of cell biology is how signaling proteins with seemingly antithetical functions, such as protein kinase (PKA) and PKC, are regulated temporally and spatially during mitoqenic stimulation. Scaffolding proteins are thought to facilitate the efficiency and specificity of substrate phosphorylation and also coordinate the actions of kinases and phosphatases in a spatiotemporal manner. The long-term goal of this research is to understand how SSeCKS, a plasma membrane-and cytoskeleton-associated PKC substrate that binds PKC, PKA, calmodulin and cyclins, helps regulate signaling crosstalk and G1-S progression in untransformed fibroblasts and epithelial cells, compared to prostate cancer cells, in which SSeCKS expression is downregulated severely. Our working hypothesis is that SSeCKS (and its orthologue, Gravin/AKAP25O) prevents PKC/PKA crosstalk: in quiescent cells, SSeCKS binds to and inhibits PKC activity whereas during G1 S progression, mitogen-activated phosphorylation of SSeCKS selectively antagonizes PKC binding; PKA, whose binding is phosphorylation insensitive, is then sequestered to "inactive" sites. With mounting data that SSeCKS has metastasis suppressor activity in prostate cancer, we envision that dissection of the PKC/PKA/SSeCKS control network will further understanding of signaling and cytoskeletal controls during mitogenesis and oncogenesis. In Aim #1, we will use site-directed mutagenesis and in vitro binding assays to identify the SSeCKS domains that bind PKC and PKA and that mediate multimerization. In Aim #2, we will examine how SSeCKS binding affects the in vitro kinase activity of PKC and PKA on specific substrates. We will also determine how the binding sites regulate PKC-and PKA-mediated G1-S signaling and cytoskeletal pathways in vivo in untransformed NIH3T3 and prostate epithelial cells, and in SSeCKS-deficient human LNCaP prostate cancer cells. We will analyze how SSeCKS affects the mitogen-and integrin-mediated cellular localization of PKC and PKA. Aim #3 will investigate how PKC-induced phosphorylation modulates SSeCKS in vivo scaffolding activity and involvement in G1-S regulation. To this end, we will map the in vivo PKC phosphorylation sites on SSeCKS under growth-promoting and oncogenic conditions using a combination of peptide microsequencing and MALDITOF mass spectrometry. The role of PKC-induced phosphorylation on SSeCKS' scaffolding/regulatory functions will be accessed using SSeCKS phospho-specific antibodies and cell lines expressing SSeCKS PKC-phosphorylation-site mutants. Given that many G1-S regulatory functions are undermined in oncogenesis, the results of the proposed research on SSeCKS should broaden our understanding of spatial and temporal controls governing cell cycle progression in normal and cancer cells.